Spectroscopic and Protein Chemical Analyses Demonstrate the Presence of C-Mannosylated Tryptophan in Intact Human RNase 2 and Its Isoforms

Abstract

Recently, the C-mannosylation of a specific tryptophan residue in RNase 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C 2 -R-mannosyltryptophan [(C 2 -Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C 2 -Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH >5 throughout these procedures, (2) demonstrated the occurrence of (C 2 -Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C 2 -Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of posttranslationally attaching carbohydrate to protein, in addition to the well-known N-and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions.

The Four Terminal Components of the Complement System AreC-Mannosylated on Multiple Tryptophan Residues

“…Edman degradation of this peptide yielded at the fourth cycle a PTH-derivative that comigrated with PTH-(C 2 -Man-)Trp obtained from human RNase 2 ( Fig. 1C), a protein whose C-mannosylation has previously been extensively characterized (5)(6)(7).…”

Glycoprotein Folding in the Endoplasmic Reticulum

“…One example is C-mannosylation of tryptophan residues within a WXXW motif. This modification also utilizes dolichol phosphate as the sugar donor (Doucey et al, 1998) and has thus far been found in RNase 2 (Loffler et al, 1996), IL-12, properdin, and other members of the complement protein family (Hofsteenge et al, 1999) (Hartmann andHofsteenge, 2000). C-mannosylation almost certainly occurs in the ER, but its timing and consequences for protein folding and activity are not known.…”

Novel forms of protein glycosylation

“…Eosinophil-deri~ed neurotoxin (El)N) is a member of thc pyrimidine-specific RNasc superfamily in vcrtcbratcs. It has the samc amino acid sequence as RNasc 2 and is also (,'-mannosylated [2]. The X-ray structure of recombinant EI)N [3] cxprcssed in E.rl~eri¢~ia (o/i was used to model thc g'-Iinkagc between nlannosc and Trp7 ( Figure 2).…”